The principal investigator has found that the product of open reading frame YNRO32C-A of S. cerevisiae functions as a posttranslational protein modifier. Orthologous open reading frames were identified in genome databases from many species, suggesting that this new protein modification is general to eukaryotes. The YNRO32C-A/RUB2 gene is not essential but is extraordinarily conserved evolutionarily. rub2 mutants exhibit a delay in the resumption of growth after stationary phase and a related phenotype of delayed germination. Rub2 exhibits scant sequence identity to ubiquitin. Mature Rub2 is generated by proteolytic processing, which generates a Tyr residue at the C-terminal site of Rub2 conjugation. This feature distinguishes Rub2 from all known ubiquitin-like proteins. In vivo targets of Rub2 conjugation, identified by mass spectrometry, include YDL223C and Sph I, a regulator of cell polarity. Using these substrates, possible functions of Rub2 in targeting proteins for degradation or for specific cellular localization will be tested. Dr. Finley proposes to identify additional physiological targets of Rub2 conjugation in S. cerevisiae, S. pombe, and in mammals. The functional significance of Rub2 conjugation will be studied through a detailed analysis of these conjugation events. In particular, he will seek to explain the defect of rub2 mutants in the resumption of growth after stationary phase or sporulation. A novel and general method is proposed to map the sites of Rub2 modification in target proteins by mass spectrometry. This will allow to generate single-residue substitutions in Rub2 modification sites, and thus to test rigorously for possible functions of Rub2 conjugation. Enzymes that mediate the formation of Rub2-protein conjugates will be identified using covalent Rub2 affinity chromatography. Enzymes that disassemble Rub2-protein conjugates will be identified by applying a rapid fluorescence polarization-based screening method to a library of all 6144 yeast ORFs expressed as GST-fusions. The complete set of yeast ORFs will also be screened robotically for putative "Rub2 receptor" proteins that may direct the fates of Rub2-protein conjugates. In summary, these studies seek to identify the key enzymes and substrates of the Rub2 pathway and thus provide an essential foundation for future studies of this novel protein modification system.